KR20210023902A - JAK3 selective inhibitor - Google Patents

Abstract

The present invention relates to a compound represented by formula (I)/(II) or a pharmaceutically acceptable salt thereof. In formula (I), Rh, Rg, Rf, m, Re, Rd, Ra, Rb, and Rc are as defined in the above description. The present invention further relates to a pharmaceutical composition comprising a compound represented by formula (I)/(II) or a pharmaceutically acceptable salt, wherein formula (I)/ in the preparation of a medicament for the treatment of inflammation such as rheumatoid arthritis It relates to the use of the compound or pharmaceutical composition represented by (II).

Description

JAK3 selective inhibitor

The present invention relates to a JAK3 selective inhibitor.

JAK kinase (JAK) and its downstream effectors, signal transducers and activators of transcription (STAT) are essential for T cell signaling. The JAK family has four members, JAK1, JAK2, JAK3 and TYK2, which bind in pairs to cytokine receptors and participate in the regulation of cytokine-mediated signaling pathways. JAK3 paired with JAK1 binds to cytokine receptors containing a γ-common chain, and is used to transmit signals such as interleukin IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Get involved. Unlike other widely expressed JAKs, JAK3 is expressed only in the hematopoietic system. Therefore, it was generally believed that selective inhibition of JAK3 could achieve a safe and effective immune effect.

Tofacitinib, an approved drug developed by Pfizer, was initially developed as a selective JAK3 inhibitor, but it was later discovered that tofacitinib also has high inhibitory activity against JAK1 and is indeed a nonselective JAK inhibitor.

Although it had an inhibitory activity similar to tofacitinib at the level of enzymatic activity, the highly selective JAK3 inhibitor NIBR3049 developed by Novartis was tofacitinib in terms of intracellular inhibitory activity against phosphorylation of the downstream substrate STAT5. It was significantly less powerful than it was.

In recent years, highly selective JAK3 inhibitors have been obtained by covalently targeting Cys909, a unique cysteine residue of JAK3, including PF-06651600 developed by Pfizer, currently under clinical Phase II study.

There is still an urgent need in the art for JAK3 selective inhibitors with high enzymatic and cellular activity.

One of the objects of the present invention is to provide a JAK3 selective inhibitor having biological activity.

In one aspect, the present invention provides a compound of formula (I) (including a stable isotope substitute thereof), or a pharmaceutically acceptable salt thereof.

here,

Rh is H or methyl, preferably H;

Rg is CH, -C-Rf or N, preferably CH;

Rf is preferably a substituent selected from methyl or halogen (eg F, Cl, Br or I);

m is 0, 1, 2 or 3, preferably 0 or 1, more preferably 0;

Re is (independently selected from -N ⁺ R _'3, wherein R' is selected from H and C ₁ -C ₆ alkyl), nitro (-NO _2), methyl (-CX _3, X = trihaloalkyl tertiary amine cation F, Cl, Br or I), halogen (such as F, Cl, Br and I), formyl (-CHO), acyl (-CO-C _1-4 alkyl), carboxyl (-COOH), cyano ( -CN), an electron-withdrawing group selected from the group consisting of a sulfonic acid group (-SO _{3 H);}

Rd is, for example, alkenyl or alkynyl having 2, 3, 4, 5 or 6 carbon atoms;

Ra, Rb and Rc are selected from the following combinations:

(1) Rb is C ₁ -C ₄ alkylene (e.g. C ₁ -C ₃ alkylene, such as methylene, ethylidene, 1,3-propylidene),

Ra and Rc are hydrogen or C ₁ -C ₆ alkyl (eg C ₁ -C ₄ alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl);

(2) Rb is C ₁ -C ₄ alkylene (e.g. C ₁ -C ₃ alkylene, such as methylene, ethylidene, 1,3-propylidene),

Ra and Rc are attached together to form C ₂ -C ₄ alkylene (eg C ₂ -C ₃ alkylene such as ethylidene, 1,3-propylidene);

(3) Ra is hydrogen or C ₁ -C ₆ alkyl (e.g. C ₁ -C ₄ alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl),

Rb and Rc together with the N atom to which they are attached form an N atom containing 5- or 6-membered saturated heterocyclic ring;

(4) Rc is hydrogen or C ₁ -C ₆ alkyl (e.g. C ₁ -C ₄ alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl),

Ra and Rb together with the N atom to which they are attached form an N atom containing 5- or 6-membered saturated heterocyclic ring.

In one aspect, the present invention provides a compound of formula II (including its stable isotope replacement), or a pharmaceutically acceptable salt thereof:

In one aspect, the present invention provides a compound of formula I (including a stable isotopic substitute thereof) or a pharmaceutically acceptable salt thereof, or a compound of formula II (including a stable isotopic substitute thereof) or a pharmaceutically acceptable salt thereof, and a pharmaceutical It provides a pharmaceutical composition comprising an acceptable carrier. Pharmaceutically acceptable carriers include inert solid fillers or excipients and sterile aqueous or organic solutions. The compound must be present in the pharmaceutical composition in an amount sufficient to provide the desired dosage. Techniques for formulating and administering the compounds disclosed in the present invention are well known to those skilled in the art, for example, Remington: the Science and Practice of Pharmacy , 19 ^th edition, Mack Publishing Company, Easton, PA (1995)].

In one aspect, the present invention relates to a compound of formula I (including a stable isotope substitute thereof) or a pharmaceutically acceptable salt thereof, a compound of formula II (a stable isotope thereof) in the manufacture of a medicament for the treatment of inflammation such as rheumatoid arthritis. Alternative) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention.

In one aspect, the present invention provides compounds of Formula I or Formula II (including stable isotopic substitutes thereof) as JAK3 selective inhibitors.

Figure 1: Results of the selectivity assay of compounds against the kinase panel;
Figures 2 to 5: Results of the cell activity assay of the compound;
6 to 8: Results of an assay for the selectivity of compounds in cells;
Figure 9: Results of evaluation of compounds in a cell washout experiment; And
10 to 12: Assay results of compounds in inhibiting the release of stimulated inflammatory cytokines

Definition of abbreviations:

Ala: Alanine

ATP: adenosine triphosphate

AUC: area under the curve

Boc: tert-butoxycarbonyl

BTK: Bruton's Tyrosine Kinase

CDI: 1,1'-carbonyldiimidazole

Cys: cysteine

DCM: dichloromethane

DIEA: N,N-diisopropylethylamine

DMF: dimethyl formamide

DMSO: dimethyl sulfoxide

EGFR: epidermal growth factor receptor

GSK3β: glycogen synthase kinase 3-β

HTRF: uniform time-resolved fluorescence

IL-2: interleukin-2

IL-6: interleukin-6

IL-15: Interleukin-15

IFN-α: interferon-α

ITK: Interleukin-2 Inducible T Cell Kinase

JAK: Janus Kinase

JAK3: Janus Kinase 3

Leu: Leucine

LPS: lipopolysaccharide

Lys: Lysine

MCP-1: mononuclear cell chemotactic protein 1

Met: methionine

PBMC: peripheral blood mononuclear cells

PBS: phosphate-buffered saline

PE: petroleum ether

PK: Pharmacokinetics

PKC: protein kinase C

RA: rheumatoid arthritis

RT (or rt): room temperature

STAT: signal transducer and activator of transcription

TFA: trifluoroacetic acid

THF: tetrahydrofuran

Val: valine

HATU: 1-[bis(dimethylamino)methylene]-1 H -1,2,3-triazolo[4,5- b ]pyridinium 3-oxide hexafluorophosphate

Pd ₂ (dba) ₃ : Tris(dibenzylideneacetone)dipalladium(0)

JohnPhos: 2-(di-tert-butylphosphino)biphenyl

RuPhos: 2-dicyclohexylphosphino-2',6'-diisopropoxybiphenyl.

Compounds were prepared according to Schemes I, II, III or IV as follows.

< Scheme I >

here,

R ₁ is selected from _{CF 3} and NO _2;

로부터 선택되고;R ₂ is

Is selected from;

로부터 선택된다;R ₃ is

Is selected from;

Reaction reagents and key conditions:

(a) i) CDI, DMF, rt, about 0.5 hours;

ii) NH ₃ (7N) in MeOH, rt, about 1 hour;

(b) ^t BuOK, THF, 0° C. to 10° C., about 45 minutes;

(c) Boc-protected piperazine or amine, DMSO, 150° C., overnight;

(d) i) TFA/DCM, rt, about 15 minutes;

ii) acryloyl chloride/DIEA, THF, H ₂ O, 0° C. to rt, about 10 minutes, or

Carboxylic acid, HATU, DIEA, DMF.

< Scheme II >

Reaction reagents and key conditions:

(a) ethyl 2-chloro-2- _{oxoacetate, Et 2} AlCl, DCM, about 2 hours, 0°C;

(b) i) CDI, DMF, rt, about 0.5 hours;

ii) NH ₃ (7N) in MeOH, rt, about 1 hour;

(c) ^t BuOK, THF, 0° C. to 10° C., about 45 minutes;

(d) Boc-protected piperazine, DMSO, 150° C., overnight;

(e) i) TFA/DCM, rt, about 15 minutes,

ii) Acryloyl chloride/DIEA, THF, H ₂ O, 0° C. to rt, about 10 minutes.

< Scheme III >

Reaction reagents and key conditions:

(a) JohnPhos or RuPhos reagent, Boc-protected piperazine, Pd ₂ (dba) ₃ , NaO ^t Bu, PhMe or DMF, microwave, 110° C., 1 hour;

(b) i) CDI, DMF, rt, about 0.5 hours;

ii) NH ₃ (7N) in MeOH, rt, about 1 hour;

(c) ^t BuOK, THF, 0° C. to 10° C., about 45 minutes (60-70%);

(d) TFA/DCM, rt, about 15 minutes;

(e) acryloyl chloride/DIEA, THF, H ₂ O, 0° C. to rt, about 10 minutes.

< Scheme IV >

Reaction reagents and key conditions:

(a) indole, _{EtMgBr in Et 2} O, THF, reflux, about 1 hour;

(b) Boc-protected amine, DIEA, DMSO, 100° C.;

(c) (i) TFA/DCM, rt, about 15 minutes;

(ii) Acryloyl chloride/DIEA, THF, H ₂ O, 0° C. to rt, about 10 minutes.

Table: Compound structure and IC ₅₀ value

The synthesis process of the compound is described in detail below by referring to Schemes I to IV above and taking compounds 5, 20, 28, 32, 34 as examples. Other compounds can be prepared in a similar manner with reference to Schemes I to IV above, which can be readily understood by one of ordinary skill in the art.

All reagents were purchased commercially and used without further purification unless otherwise specified. The solvent was re-evaporated before use. The reaction was monitored by a thin layer silica gel plate (TLC, GF254, 60-F250, 0.2 mm, Yantai Jiangyou silica gel thin layer chromatography). Flash column chromatography was performed using Puke silica gel (ZCX-II, 200-300 mesh). NMR spectra were recorded on a Bruker ADVANCE 400 ( ¹ H: 400 MHz; ¹³ C: 100 MHz) or a Bruker Advance 500 ( ¹ H: 500 MHz; ¹³ C: 125 MHz) nuclear magnetic resonance instrument. I did. TMS was used as an internal standard, and the peak shapes were described as s (single line), d (doublet), t (triplet) and m (multiples). High-resolution mass spectrometry (HRMS) was performed using an ABI Q-star Elite high-resolution mass spectrometer; The purity of the final product was detected by a high performance liquid (HPLC) Agilent 1260 series chromatograph (Agilent PN959990-902 Eclipse Plus C18 (250 mm*4.6 mm) chromatography column), and the detection was It was measured at 254 nm.

Preparation of compound 5

3-(5-(4-acryloylpiperazin-1-yl)-2-(trifluoromethyl)phenyl)-4-(1 H -indol-3-yl)-1 H -pyrrole-2, Synthesis of 5-dione (compound 5)

Step 1: 2-(5-fluoro-2-(trifluoromethyl)phenyl)acetamide (compound 38a)

CDI (1.0 g, 4.5 mmol) was added batchwise to a solution of (5-fluoro-2-(trifluoromethyl)phenyl)acetic acid in 4 mL of DMF. After stirring for 0.5 h at room temperature (rt), NH ₃ (3.6 mL, 7 N in methanol solution) was added dropwise and stirred at room temperature for another 1 hour. The solvent was evaporated and the product was extracted with water and ethyl acetate (2 x 120 mL). The organic phase was washed with saturated brine (2 x 40 mL), collected, _{dried over anhydrous Na 2} SO ₄ , concentrated, then separated and purified by column chromatography to give a white solid (0.73 g) of 73% It was obtained in the yield of.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 7.75 (dd, J = 8.7, 5.6 Hz, 1H), 7.52 (s, 1H), 7.32 (m, 2H), 7.03 (s, 1H), 3.66 (s , 2H); MS (ESI) m/z 222.0 (M+H) ⁺ .

Step 2: 3-(5-fluoro-2-(trifluoromethyl)phenyl)-4-(1 H -indol-3-yl)-1 H -pyrrole-2,5-dione (compound 40a)

At 0° C., ^t BuOK (5.5 mL, 1 M in THF) was added to a solution of compound 38a (0.30 g, 1.3 mmol) and compound 39 (0.41 g, 2.0 mmol) in anhydrous THF (8.0 mL). The solution was stirred at 10° C. for 45 minutes. After completion of the reaction (detected by TLC), HCl (5N) was added to adjust the pH to 6, the solvent was removed, the mixture was extracted with ethyl acetate (2 x 120 mL), and the organic phase was saturated brine ( 2 x 40 mL), collected, _{dried over anhydrous Na 2} SO ₄ , concentrated, then separated and purified by column chromatography to give a yellow solid (0.35 g) in 66% yield.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 11.99 (s, 1H), 11.23 (s, 1H), 8.02 (d, J = 3.0 Hz, 1H), 7.97 (dd, J = 8.9, 5.4 Hz, 1H ), 7.60-7.52 (m, 1H), 7.49-7.38 (m, 2H), 7.07 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 6.75 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H ), 6.46 (d, J = 8.3 Hz, 1H).

¹³ CNMR (101 MHz, DMSO) δ 172.48, 172.01, 165.30, 162.80, 137.08, 136.25, 132.67, 126.18, 125.48, 125.25, 122.91, 120.95, 120.56, 120.19, 119.96, 117.31, 117.09, 112.94, 105.14.

MS (ESI) m/z 375.1 (M+H) ⁺ .

Step 3: Tert-Butyl 4-(3-(4-(1 H -indol-3-yl)-2,5-dioxo-2,5-dihydro-1 H -pyrrol-3-yl)-4 -(Trifluoromethyl)phenyl)piperazine-1-carboxylate (compound 41a)

1-Boc-piperazine (0.44 g, 2.4 mmol) was added to a solution of 40a (0.3 g, 0.55 mmol) in DMSO (2.0 mL) and the mixture was refluxed at 150° C. overnight. After completion of the reaction (detected by TLC), the mixture was cooled to room temperature, extracted with ethyl acetate (2 x 120 mL), and the organic phase was washed with saturated brine (2 x 40 mL), collected, and anhydrous Dried over Na ₂ SO ₄ , concentrated, then separated and purified by column chromatography to give a yellow solid (0.35 g) in 66% yield.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 11.88 (s, 1H), 11.10 (s, 1H), 7.95 (d, J = 2.9 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.18-7.10 (m, 1H), 7.09-6.99 (m, 1H), 6.92 (d, J = 2.6 Hz, 1H), 6.72 (dd, J = 8.2, 7.0 Hz, 1H), 6.61 (d, J = 8.2 Hz, 1H), 3.34-3.22 (m, 5H), 3.24-3.09 (m, 4H), 1.38 (s, 9H).

¹³ CNMR (101 MHz, DMSO) δ 172.85, 172.40, 154.34, 152.82, 136.97, 135.64, 132.09, 131.86, 131.85, 128.70, 128.42, 126.50, 126.50, 125.39, 125.38, 122.69, 121.37, 120.70, 112.6497. , 105.55, 79.57, 49.00, 47.32, 30.63, 29.52, 28.55.

MS (ESI) m/z 541.2 (M+H) ⁺ .

Step 4: 3-(5-(4-acryloylpiperazin-1-yl)-2-(trifluoromethyl)-phenyl)-4-(1 H -indol-3-yl)-1 H- Pyrrole-2,5-dione (Compound 5)

Trifluoroacetic acid (TFA) (2.0 mL) was added to a solution of compound 41a (0.10 g, 0.18 mmol) in DCM (2.0 mL) and the mixture was stirred at room temperature for 15 minutes. After completion of the reaction (detected by TLC), TFA and DCM were evaporated and the residue was dried and used in the next step without further purification. The residue was dissolved in a mixture of THF (2.0 mL) and water (1 drop), then DIEA (0.10 mL, 0.36 mmol) and acryloyl chloride (24 μL, 0.27 mmol) were added. The ice bath was removed and the resulting solution was stirred at room temperature for 10 minutes. After completion of the reaction (detected by TLC), the mixture was extracted with ethyl acetate (2 x 120 mL), the organic phase was washed with saturated brine (2 x 40 mL), collected, and dried over anhydrous Na ₂ SO ₄ After concentration, separation and purification by column chromatography gave a yellow solid (73 mg) in 82% yield.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 11.88 (d, J = 3.0 Hz, 1H), 11.11 (s, 1H), 7.96 (d, J = 2.6 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.18-7.10 (m, 1H), 7.08-7.00 (m, 1H), 6.96 (d, J = 2.5 Hz, 1H), 6.76 (m , 2H), 6.65 (d, J = 8.3 Hz, 1H), 6.11 (dd, J = 16.6, 2.4 Hz, 1H), 5.68 (dd, J = 10.4, 2.4 Hz, 1H), 3.66-3.46 (m, 4H), 3.23 (m, 4H).

¹³ CNMR (101 MHz, DMSO) δ 172.64, 172.20, 164.62, 152.49, 136.73, 135.43, 131.85, 131.65, 128.49, 128.34, 127.86, 126.28, 125.18, 123.57, 122.50, 121.15, 120.51, 117.42, 105.54.54 , 47.58, 47.01, 44.37, 40.90.

HRMS (ESI) m/z calculated for C ₂₆ H ₂₁ F ₃ N ₄ O ₃ [M+H] ^{+: 495.1566.} Found, 495.1578.

Preparation of compound 20

3-(5-(4-acryloylpiperazin-1-yl)-2-(trifluoromethyl)phenyl)-4-(6-fluoro-1 H -indol-3-yl)-1 H -Synthesis of pyrrole-2,5-dione (compound 20)

Step 1: Ethyl 2-(6-Fluoro-1 H -indol-3-yl)-2-oxoacetate (Compound 43a)

To a solution of compound 42a (0.50 g, 3.7 mmol) in DCM (40 mL) was added 5.6 mL of Et ₂ AlCl (1 M in hexane) under an ice bath. The mixture was stirred at 0° C. for 30 minutes. Ethyl oxalyl monochloride (0.61 mL, 5.5 mmol) was added dropwise to this solution at 0°C. The resulting solution was stirred at 0° C. for 2 h, and then after the reaction was complete (detected by TLC) ice water was added to quench the reaction. The solvent was evaporated and the product was extracted with water and ethyl acetate (3 x 50 mL). The organic phase was washed with saturated brine (2 x 30 mL), collected, _{dried over anhydrous Na 2} SO ₄ , concentrated, then separated and purified by column chromatography to give an off-white solid (0.38 g) of 50% It was obtained in the yield of.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 12.41 (s, 1H), 8.44 (s, 1H), 8.14 (dd, J = 8.7, 5.5 Hz, 1H), 7.35 (dd, J = 9.5, 2.4 Hz , 1H), 7.13 (ddd, J = 9.8, 8.7, 2.4 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H).

MS (ESI) m/z 236.1 (M+H) ⁺ .

Step 2: 3-(6-fluoro-1 H -indol-3-yl)-4-(5-fluoro-2-(trifluoromethyl)phenyl)-1 H -pyrrole-2,5-dione (Compound 44a)

At 0° C., ^t BuOK (4.0 mL, 1 M in THF) was added to a solution of compound 38a (0.19 g, 0.85 mmol) and compound 43a (0.30 g, 1.3 mmol) in anhydrous THF (4.0 mL). The solution was stirred at 10° C. for 1 h. After completion of the reaction (detected by TLC), HCl (5N) was added to adjust the pH to 6, the solvent was removed, the mixture was extracted with ethyl acetate (3 x 50 mL), and the organic phase was saturated brine ( 2 x 20 mL), collected, _{dried over anhydrous Na 2} SO ₄ , concentrated, then separated and purified by column chromatography to give a yellow solid (0.25 g) in 75% yield.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 12.06 (s, 1H), 11.26 (s, 1H), 8.03 (d, J = 3.1 Hz, 1H), 8.00 (dd, J = 8.9, 5.4 Hz, 1H ), 7.60 (m, 1H), 7.49 (dd, J = 9.2, 2.7 Hz, 1H), 7.42 (dd, J = 8.9, 4.8 Hz, 1H), 6.94 (m, 1H), 6.14 (dd, J = 11.1, 2.5 Hz, 1H).

MS (ESI) m/z 393.0 (M+H) ⁺ .

Step 3: tert-butyl 4-(3-(4-(6-fluoro-1 H -indol-3-yl)-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3 -Yl)-4-(trifluoromethyl)phenyl)piperazine-1-carboxylate (compound 45a)

1-Boc-piperazine (0.44 g, 2.4 mmol) was added to a solution of compound 44a (0.3 g, 0.55 mmol) in DMSO (2.0 mL) and the mixture was refluxed at 150° C. overnight. After completion of the reaction (detected by TLC), the mixture was cooled to room temperature, extracted with ethyl acetate (2 x 120 mL), and the organic phase was washed with saturated brine (2 x 40 mL), collected, and anhydrous Dried over Na ₂ SO ₄ , concentrated, then separated and purified by column chromatography to give a yellow solid (0.35 g) in 30% yield.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 11.87 (s, 1H), 11.13 (s, 1H), 7.89 (s, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.17 (dd, J = 9.5, 2.3 Hz, 1H), 7.13 (dd, J = 8.8, 2.6 Hz, 1H), 6.94 (d, J = 2.6 Hz, 1H), 6.70-6.57 (m, 2H), 3.33-3.25 (m, 4H), 3.22 (m, 4H), 1.39 (s, 9H).

MS (ESI) m/z 559.2 (M+H) ⁺ .

Step 4: 3-(5-(4-acryloylpiperazin-1-yl)-2-(trifluoromethyl)-phenyl)-4-(6-fluoro-1 H -indol-3-yl ) -1 H - pyrrole-2,5-dione (compound 20)

Trifluoroacetic acid (TFA) (2.0 mL) was added to a solution of compound 45a (0.10 g, 0.18 mmol) in DCM (2.0 mL) and the mixture was stirred at room temperature for 15 minutes. After completion of the reaction (detected by TLC), TFA and DCM were evaporated and the residue was dried and used in the next step without further purification. The residue was dissolved in a mixture of THF (2.0 mL) and water (1 drop), then DIEA (0.10 mL, 0.36 mmol) and acryloyl chloride (24 μL, 0.27 mmol) were added. The ice bath was removed and the resulting solution was stirred at room temperature for 10 minutes. After completion of the reaction (detected by TLC), the mixture was extracted with ethyl acetate (2 x 120 mL), the organic phase was washed with saturated brine (2 x 40 mL), collected, and dried over anhydrous Na ₂ SO ₄ After concentration, separation and purification by column chromatography gave a yellow solid (73 mg) in 80% yield.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 11.86 (s, 1H), 11.12 (s, 1H), 7.87 (s, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.21-7.08 (m , 2H), 6.97 (d, J = 2.3 Hz, 1H), 6.78 (m, 1H), 6.69 (m, 1H), 6.62 (m, 1H), 6.11 (dd, J = 16.7, 2.3 Hz, 1H) , 5.68 (dd, J=10.4, 2.3 Hz, 1H), 3.55 (m, 4H), 3.24 (m, 4H).

¹³ CNMR (101 MHz, DMSO-d ₆ ) δ 172.70, 172.30, 164.88, 158.20, 152.82, 137.14, 137.02, 135.41, 132.57, 129.48, 128.63, 128.05, 122.60, 122.51, 122.17, 117.72, 114.79, 109.16, 108.92 105.65, 98.84, 98.59, 47.75, 47.16, 44.65, 41.14.

HRMS (ESI) m/z calculated for C ₂₆ H ₂₀ F ₄ N ₄ O ₃ [M+H] ^{+: 513.1472.} Found, 513.1479. Purity: 99.2%.

Preparation of compound 28

3-(5-(4-acryloylpiperazin-1-yl)-2-methoxyphenyl)-4-(1 H -indol-3-yl)-1 H -pyrrole-2,5-dione ( Synthesis of compound 28)

Step 1: 2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-methoxyphenyl)acetic acid (compound 47d)

Compound 46d (0.52 g, 2.0 mmol), 1-Boc-piperazine (0.49 g, 2.6 mmol), sodium tert-butoxide (0.59 g, 2.6 mmol), 2-(ditert-butylphosphino)biphenyl ( JohnPhos, 0.16 g, 0.41 mmol), and Pd ₂ (dba) ₃ (0.19 g, 0.20 mmol) were dissolved in anhydrous PhMe (15 mL) in a microwave vial and purged with argon gas to remove oxygen. The vial was capped and heated to 110° C. for 1 hour. After cooling to room temperature and completion of the reaction (detected by TLC), the mixture was filtered through diatomaceous earth, the filtrate was adjusted to pH 5 and extracted with ethyl acetate (3 x 100 mL). The organic phase was washed with saturated brine (2 x 40 mL), collected, _{dried over anhydrous Na 2} SO ₄ , concentrated, then separated and purified by column chromatography to give a white solid (0.35 g) to 49% It was obtained in the yield of.

¹ HNMR (500 MHz, DMSO-d ₆ ) δ 6.87-6.84 (m, 1H), 6.84 (s, 1H), 6.82 (d, J = 2.8 Hz, 1H), 3.68 (s, 3H), 3.45 (s , 2H), 3.45-3.41 (m, 4H), 2.95-2.93 (m, 4H), 1.42 (s, 9H).

¹³ CNMR (126 MHz, DMSO) δ 173.04, 154.32, 152.01, 145.32, 124.49, 121.12, 116.45, 111.74, 79.40, 56.13, 50.34, 36.23, 28.53.

MS/ESI 351.2 (M+1) ⁺ .

Step 2: tert-Butyl 4-(3-(2-amino-2-oxoethyl)-4-methoxyphenyl)piperazine-1-carboxylate (compound 48d)

CDI (0.29 g, 1.29 mmol) was added batchwise to a solution of compound 47d (0.30 g, 0.86 mmol) in 4.0 mL of DMF. After stirring at room temperature for 0.5 h, NH ₃ (0.6 mL, 7 N in methanol solution) was added and stirred at room temperature for another 1 h. The solvent was evaporated and the product was extracted with water and ethyl acetate (2 x 120 mL). The organic phase was washed with saturated brine (2 x 40 mL), collected, _{dried over anhydrous Na 2} SO ₄ , concentrated, then separated and purified by column chromatography to give a white solid (0.73 g) of 73% It was obtained in the yield of.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 7.75 (dd, J = 8.7, 5.6 Hz, 1H), 7.52 (s, 1H), 7.32 (m, 2H), 7.03 (s, 1H), 3.66 (s , 2H);

MS (ESI) m/z 222.0 (M+H) ⁺ .

Step 3: tert-butyl 4-(3-(4-(1 H -indol-3-yl)-2,5-dioxo-2,5-dihydro-1 H -pyrrol-3-yl)-4 -Methoxyphenyl)piperazine-1-carboxylate (Compound 49d)

At 0° C., ^t BuOK (2.2 mL, 1 M in THF) was slowly added to a solution of compound 48d (0.2 g, 0.57 mmol) and compound 38a (0.17 g, 0.85 mmol) in anhydrous THF (4.0 mL). The solution was stirred at 10° C. for 1 h. After completion of the reaction (detected by TLC), HCl (5N) was added to adjust the pH to 5, the solvent was removed, the mixture was extracted with ethyl acetate (3 x 40 mL), and the organic phase was saturated brine ( 2 x 20 mL), collected, _{dried over anhydrous Na 2} SO ₄ and concentrated to give intermediate 49d.

MS (ESI) m/z 503.2 (M+H) ⁺ .

Step 4: 3- (1 H - indol-3-yl) -4- (2-methoxy-5- (piperazin-1-yl) phenyl) -1 H - pyrrole-2,5-dione (Compound 50d )

Trifluoroacetic acid (TFA) (2.0 mL) was added to a solution of compound 49d (0.12 g, 0.24 mmol) in DCM (2.0 mL) and the mixture was stirred at room temperature for 15 minutes. After completion of the reaction (detected by TLC), TFA and DCM are evaporated, the mixture is extracted with water and ethyl acetate (2 x 60 mL), the organic phase is washed with saturated brine (2 x 30 mL) and collected , Dried over anhydrous Na ₂ SO ₄ , concentrated, then separated and purified by column chromatography to give a yellow solid (0.15 g) in 52% yield.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 11.80 (s, 1H), 7.91 (s, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.02 (ddd, J = 8.2, 7.0, 1.1 Hz , 1H), 6.95 (dd, J = 9.0, 3.0 Hz, 1H), 6.88 (d, J = 9.1 Hz, 1H), 6.78 (d, J = 2.9 Hz, 1H), 6.65 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 6.51-6.41 (m, 1H), 3.26 (s, 3H), 2.89-2.81 (m, 4H), 2.80-2.78 (m, 4H).

¹³ CNMR (101 MHz, DMSO) δ 173.08, 172.67, 152.06, 145.93, 136.87, 134.69, 130.87, 128.29, 125.38, 122.39, 121.29, 121.12, 120.23, 119.96, 118.58, 113.08, 112.38, 106.29, 55.97, 50. .

MS/ESI 503.2 (M+1) ⁺ .

Step 5: 3-(5-(4-acryloylpiperazin-1-yl)-2-methoxyphenyl)-4-(1 H -indol-3-yl)-1 H -pyrrole-2,5 -Dion (Compound 28)

Compound 50d (0.12 g, 0.24 mmol) was dissolved in a mixture of THF (2.0 mL) and water (1 drop), then DIEA (0.16 mL, 0.96 mmol) and acryloyl chloride (30 μL, 0.36 mmol) were added. I did. The ice bath was removed and the resulting solution was stirred at room temperature for 10 minutes. After completion of the reaction (detected by TLC), the mixture was extracted with ethyl acetate (2 x 60 mL). The organic phase was washed with saturated brine (2 x 30 mL), collected, _{dried over anhydrous Na 2} SO ₄ , concentrated, then separated and purified by column chromatography to give a yellow solid (90 mg) 82% It was obtained in the yield of.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 11.81 (s, 1H), 10.93 (s, 1H), 7.93 (d, J = 2.8 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.07-6.98 (m, 2H), 6.92 (d, J = 9.0 Hz, 1H), 6.84 (d, J = 2.9 Hz, 1H), 6.80 (dd, J = 16.7, 10.5 Hz, 1H), 6.66 (t , J = 7.3 Hz, 1H), 6.45 (d, J = 8.1 Hz, 1H), 6.10 (dd, J = 16.7, 2.4 Hz, 1H), 5.68 (dd, J = 10.4, 2.4 Hz, 1H), 3.59 (m, 4H), 3.29 (s, 3H), 2.91 (m, 4H).

¹³ CNMR (101 MHz, DMSO-d ₆ ) δ 173.05, 172.62, 164.74, 152.47, 144.97, 136.88, 134.75, 130.96, 128.69, 128.10, 127.93, 125.33, 122.42, 121.25, 121.20, 120.60, 120.26, 119.13, 113. 112.42, 106.23, 55.98, 50.83, 50.22, 45.27, 41.73.

HRMS (ESI) m/z calculated for C ₂₆ H ₂₄ N ₄ O ₄ [M+H] ^{+: 457.1798.} Found, 457.1794.

Preparation of compound 32

Step 1: 2-(5-fluoro-2-(nitro)phenyl)acetamide (compound 38b)

CDI (1.2 g, 7.5 mmol) was added batchwise to a solution of 5-fluoro-2-(nitro)phenylacetic acid (1.0 g, 5.0 mmol) in 4.0 mL of DMF. After stirring at room temperature for 0.5 h, NH ₃ (3.5 mL, 7 N in methanol solution) was added dropwise and stirred at room temperature for another 1 h. The solvent was evaporated and the product was extracted with water and ethyl acetate (2 x 120 mL). The organic phase was washed with saturated brine (2 x 40 mL), collected, _{dried over anhydrous Na 2} SO ₄ , concentrated, then separated and purified by column chromatography to give a white solid (0.70 g) to 70% It was obtained in the yield of.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.11 (dd, J = 9.0, 5.2 Hz, 1H), 7.54 (s, 1H), 7.46-7.29 (m, 2H), 7.02 (s, 1H), 3.88 (s, 2H).

MS (ESI) m/z 199.1 (M+H) ⁺ .

Step 2: 3- (5-fluoro-2- (nitro) phenyl) -4- (1H- indol-3-yl) -1 H - pyrrole-2,5-dione (Compound 40b)

At 0° C., ^t BuOK (7.5 mL, 1 M in THF) was slowly added to a solution of compound 38b (0.30 g, 1.5 mmol) and compound 39 (0.45 g, 2.2 mmol) in anhydrous THF (15 mL). The solution was stirred at 10° C. for 45 minutes. After completion of the reaction (detected by TLC), HCl (5N) was added to adjust the pH to 6, the solvent was removed, the mixture was extracted with ethyl acetate (2 x 120 mL), and the organic phase was saturated brine ( 2 x 40 mL), collected, _{dried over anhydrous Na 2} SO ₄ and concentrated to give 0.30 g of a yellow solid in 72% yield.

MS (ESI) m/z 352.2 (M+H) ⁺ .

Step 3: Tert-Butyl 4-(3-(4-(1 H -indol-3-yl)-2,5-dioxo-2,5-dihydro-1 H -pyrrol-3-yl)-4 -(Nitro)phenyl)piperazine-1-carboxylate (compound 41b)

1-Boc-piperazine (0.64 g, 3.4 mmol) was added to a solution of 40b (0.3 g, 0.85 mmol) in DMSO (2.0 mL) and the mixture was refluxed at 150° C. overnight. After completion of the reaction (detected by TLC), the mixture was cooled to room temperature, extracted with ethyl acetate (2 x 120 mL), and the organic phase was washed with saturated brine (2 x 40 mL), collected, and anhydrous Dried over Na ₂ SO ₄ , concentrated, then separated and purified by column chromatography to give a yellow solid (0.27 g) in 62% yield.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.94 (s, 1H), 11.12 (s, 1H), 8.17 (d, J = 9.4 Hz, 1H), 8.03 (s, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.16-6.94 (m, 2H), 6.74 (t, J = 7.6 Hz, 1H), 6.65-6.49 (m, 2H), 3.14 (d, J = 48.7 Hz, 4H), 2.97 (s, 4H), 1.37 (s, 9H).

¹³ C NMR (101 MHz, DMSO) δ 172.88, 171.47, 154.25, 153.43, 137.86, 137.08, 132.84, 131.86, 129.73, 129.47, 127.83, 124.65, 122.61, 121.04, 120.56, 116.08, 114.45, 7961, 104. 60.29, 57.90, 46.77, 28.52.

MS (ESI) m/z 518.4 (M+H) ⁺ .

Step 4: 3-(5-(4-acryloylpiperazin-1-yl)-2-(nitro)phenyl)-4-(1 H -indol-3-yl)-1 H -pyrrole-2, 5-dione (compound 32)

Trifluoroacetic acid (TFA) (2.0 mL) was added to a solution of compound 41b (0.10 g, 0.19 mmol) in DCM (2.0 mL) and the mixture was stirred at room temperature for 15 minutes. After completion of the reaction (detected by TLC), TFA and DCM were evaporated and the residue was dried and used in the next step without further purification. The residue was dissolved in a mixture of THF (2.0 mL) and water (1 drop), and DIEA (70 μL, 0.38 mmol) and acryloyl chloride (26 μL, 0.28 mmol) were added. The ice bath was removed and the resulting solution was stirred at room temperature for 10 minutes. After completion of the reaction (detected by TLC), the mixture was extracted with ethyl acetate (2 x 120 mL), the organic phase was washed with saturated brine (2 x 40 mL), collected, and dried over anhydrous Na ₂ SO ₄ And concentrated, then separated by column chromatography and purified to give a yellow solid (70 mg) in a yield of 78%.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.96 (s, 1H), 11.14 (s, 1H), 8.18 (d, J = 9.3 Hz, 1H), 8.03 (s, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.07 (d, J = 9.6 Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H), 6.82-6.71 (m, 1H), 6.70-6.62 (m, 1H), 6.60-6.50 (m, 2H), 6.08 (d, J = 16.6 Hz, 1H), 5.66 (d, J = 10.5 Hz, 1H), 3.26 (m, 4H), 3.10 (m, 4H).

¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 173.17, 171.51, 164.85, 153.40, 138.29, 137.06, 133.13, 131.87, 129.76, 128.52, 128.09, 127.85, 124.69, 122.67, 121.05, 120.60, 115.95, 114.10 , 104.46, 80.45, 47.53, 46.29, 44.15, 43.52.

HRMS (ESI) m/z calculated for C ₂₅ H ₂₁ N ₅ O ₅ [M+H] ^{+: 472.1543.} Found, 472.1539.

Preparation of compound 34

Synthesis of 3-((1-acryloylpiperidin-4-yl)amino)-4-(1 H -indol-3-yl)-1 H -pyrrole-2,5-dione (Compound 34)

Step 1: 3-bromo-4-(1 H -indol-3-yl)-1 H -pyrrole-2,5-dione (compound 52)

In a two-necked flask equipped with a dropping funnel under argon, indole (0.3 g, 1.2 mmol) was dissolved in anhydrous THF (8.0 mL). A solution of ethyl magnesium bromide in Et ₂ O (1.57 mL, 4.7 mmol) was added dropwise to the mixture, which was then heated under reflux for 2 hours. After cooling to room temperature, a solution of 3,4-dibromo-1 H -pyrrole-2,5-dione (compound 51, 0.55 g, 4.7 mmol) in THF was added dropwise over about 1 h. Then, the reaction mixture was stirred at room temperature for 1 h. After completion of the reaction (detected by TLC), the mixture was then hydrolyzed to pH = 9 with aqueous HCl solution. After addition of saturated aqueous NH ₄ Cl solution, the aqueous phase was extracted with ethyl acetate (2 x 60 mL). The organic phase was washed with saturated brine (2 x 30 mL), collected, _{dried over anhydrous Na 2} SO ₄ , concentrated, then separated and purified by column chromatography to give a yellow solid (0.28 g) 82% It was obtained in the yield of.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 12.10 (s, 1H), 11.35 (s, 1H), 8.03 (d, J = 2.9 Hz, 1H), 7.89 (dt, J = 8.1, 1.0 Hz, 1H ), 7.51 (dt, J = 8.1, 1.0 Hz, 1H), 7.22 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 7.14 (ddd, J = 8.1, 7.1, 1.2 Hz, 1H).

¹³ CNMR (101 MHz, DMSO) δ 170.75, 167.99, 138.54, 137.01, 131.54, 125.05, 122.95, 122.77, 120.92, 115.13, 112.84, 104.25.

MS/ESI 291.0 (M+1) ⁺ .

Step 2: tert-butyl 4-((4-(1 H -indol-3-yl)-2,5-dioxo-2,5-dihydro-1 H -pyrrol-3-yl)amino)piperi Dean-1-carboxylate (compound 53)

Compound 52 (0.13 g, 0.45 mmol) and Boc protected piperidin-4-amine (0.18 g, 0.89 mmol) were dissolved in DMSO (1.5 mL), followed by DIEA (0.15 mL, 0.89 mmol). The mixture was heated at 126° C. overnight. After cooling to room temperature and completion of the reaction (detected by TLC), the mixture was extracted with water and ethyl acetate. The organic phase was washed with saturated brine (2 x 20 mL), collected, _{dried over anhydrous Na 2} SO ₄ , concentrated, then separated and purified by column chromatography to give a yellow solid (0.11 g) to 60% It was obtained in the yield of.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 11.21 (d, J = 2.5 Hz, 1H), 10.34 (s, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.37-7.27 (m, 2H) ), 7.14-7.06 (m, 1H), 7.04-6.95 (m, 1H), 6.86 (d, J = 9.0 Hz, 1H), 3.67 (m, 2H), 3.43 (m, 1H), 1.97 (m, 2H), 1.47 (m, 2H), 1.31 (s, 9H), 1.26 (m, 2H). ¹³ CNMR (101 MHz, DMSO).

¹³ CNMR (101 MHz, DMSO) δ 173.82, 169.57, 154.19, 143.23, 136.10, 128.71, 126.48, 121.70, 119.91, 119.37, 112.05, 104.59, 100.05, 93.48, 79.22, 50.31, 32.01, 28.52.

MS/ESI 410.1 (M+1) ⁺ .

Step 3: 3-((1-acryloylpiperidin-4-yl)amino)-4-(1 H -indol-3-yl)-1 H -pyrrole-2,5-dione (Compound 34)

TFA (2.0 mL) was added to a solution of compound 53 (0.10 g, 0.18 mmol) in DCM (2.0 mL) and the mixture was stirred at room temperature for 15 min. After completion of the reaction (detected by TLC), TFA and DCM were evaporated and the residue was dried and used in the next step without further purification. The residue was dissolved in a mixture of THF (2.0 mL) and water (1 drop), then DIEA (0.10 mL, 0.36 mmol) and acryloyl chloride (24 μL, 0.27 mmol) were added. The ice bath was removed and the resulting solution was stirred at room temperature for 10 minutes. After completion of the reaction (detected by TLC), the mixture was extracted with ethyl acetate (2 x 120 mL), the organic phase was washed with saturated brine (2 x 40 mL), collected, and dried over anhydrous Na ₂ SO ₄ And concentrated, then separated by column chromatography and purified to give a yellow solid (80 mg) in 90% yield.

¹ HNMR (400 MHz, DMSO-d ₆ ) δ 11.23 (s, 1H), 10.43 (s, 1H), 7.54-7.20 (m, 3H), 7.09 (d, J = 7.1 Hz, 1H), 7.01 ( t, J = 7.0 Hz, 1H), 6.64 (d, J = 7.5 Hz, 1H), 5.75 (t, J = 14.6 Hz, 1H), 5.27 (d, J = 7.8 Hz, 1H), 3.87 (d, J = 12.2 Hz, 1H), 2.93 (m, 1H), 2.82 (m, 1H), 2.56 (m, 1H), 1.75 (m, 1H), 1.57 (m, 2H), 0.84 (m, 1H) .

¹³ CNMR (101 MHz, DMSO) δ 173.79, 169.60, 164.78, 142.34, 136.13, 128.48, 128.46, 127.84, 127.35, 126.68, 121.84, 119.96, 119.59, 112.24, 104.14, 50.10, 49.39, 41.81, 30.

HRMS (ESI) m/z calculated for C ₂₀ H ₂₀ N ₄ O ₃ [M+H] ^{+: 365.1535;} Found, 365.1541. Purity: 99.3%.

Biological activity test

(1) In vitro enzyme assay

The in vitro enzyme assay _{was performed under conditions of K m} ATP (0.6 micromolar) and high concentration ATP (1 mmol). The results of the in vitro enzyme assay are listed in the table above.

The procedure for the in vitro enzyme assay is as follows: Kinase was obtained from Carna Biosciences. The enzyme activity of JAK3 was ^{evaluated using the HTRF ®} KinEase™ assay using separate ATP concentrations at Km and 1 mM. The ATP Kinase Enzymatic Assay was ^{performed according to the protocol specified by the HTRF ®} KinEase™ Assay Guidelines (Cisbio Bioassays).

(2) Selectivity test

To evaluate the selectivity of compound 32 to the kinase panel, 50 representative kinases were selected for the preliminary selectivity assay, and the results are shown in FIG. 1.

Compound 32 was shown to have high selectivity. As a result of testing at 1 μM on the kinase panel, most of the kinases showed 50% or less inhibition, and only the three kinases PKCα, PKCγ and GSK3β showed greater than 50% inhibition, consistent with the selectivity results of NIBR3049. Compound 32 also exhibited high selectivity within the JAK family and among 10 other kinases with cysteine at a position comparable to Cys909, which compound 32 could be used as a small molecule probe for the study of JAK3 function and JAK-STAT signaling pathway. Indicates that there is.

(3) Cell activity assay

To evaluate the cellular activity of compound 32, the ability of compound 32 to inhibit phosphorylation of the downstream substrate STAT5 in cells was detected.

Mouse T cells (CTLL-2 cells) removed growth factors and maintained starvation overnight. The cells were then incubated for 2 hours at 37° C. with the specified concentration of the compound (JAK3 inhibitor or DMSO). After stimulation with 500 ng/mL IL-2 or 500 ng/mL IL-5 (R&D Systems) for 30 minutes, cells were collected and lysed in cell lysis buffer containing protease and phosphatase inhibitors. . Subsequently, it was separated by SDS/PAGE electrophoresis, transferred to a nitrocellulose membrane, and then subjected to Western blotting analysis. Phospho-STAT5, STAT5 and β-actin (all antibodies are from Cell Signaling Technologies) were blotted separately with specific antibodies. The results are shown in FIGS. 2 and 3. EC ₅₀ values were calculated by quantitative stripe gray analysis using GraphPad Prism software.

In CTLL-2, IL-2-induced STAT5 phosphorylation was almost completely inhibited by _{600 nanomolar (EC 50 =305 nanomolar) of compound 32.} In comparison, 6000 nanomolar was required in the cells treated with compound NIBR3049 to completely inhibit STAT5 activation (EC ₅₀ = 1999 nanomolar). Compound 32 was more sensitive to inhibiting IL-15-induced STAT5 phosphorylation (EC ₅₀ =141 nanomolar).

Similarly, in human peripheral blood mononuclear cells (PBMC), compared to NIBR3049, compound 32 also showed higher inhibitory activity against IL-2 and IL-15-induced STAT5 phosphorylation. The results are shown in FIGS. 4 and 5.

Method: After thawing, PBMC (purchased from AllCells) was resuspended overnight in RPMI-1640 containing 10% FBS and then incubated for 2 h with the specified concentration of JAK3 inhibitor or DMSO. IL-2 (500 ng/mL, R&D Systems), IL-15 (500 ng/mL, R&D Systems), IL-6 (600 ng/mL, R&D Systems), or IFN-α ( After stimulation for 30 minutes at 400 ng/mL, R&D Systems), cells were collected and lysed in cell lysis buffer containing protease and phosphatase inhibitors. Subsequently, it was separated by SDS/PAGE electrophoresis, transferred to a nitrocellulose membrane, and then subjected to Western blotting analysis. Phospho-STAT5, Phospho-STAT3, and Phospho-STAT1 (all from Cell Signaling Technologies) were blotted separately with specific antibodies. β-actin was blotted with the same loading. EC ₅₀ values were calculated by quantitative stripe gray analysis using GraphPad Prism software.

(4) Cell selectivity assay

We further characterized the cellular selectivity of Compound 32 in PBMCs immediately after stimulation with different cytokines (IL-15, IL-6 or IFN-α) by detecting the inhibitory effect of the compound on the phosphorylation of the downstream substrate of JAK. The method is as described in section (4), and the results are shown in Figures 6, 7 and 8.

Of these cytokines, only signaling through IL-15 is dependent on JAK3. Signaling through IL-6 is dependent on JAK1, JAK2 and TYK2, and signaling through IFN-α is exclusively related to JAK1 and TYK2. Compound 32 effectively eliminated the phosphorylation of STAT5 at a concentration of 300 nM. In comparison, in the I-6 and IFN-α signaling pathways, only partial inhibition of STAT3 phosphorylation and STAT1 phosphorylation was observed even at doses as high as 10 μM. Compared to compound NIBR3049, compound 32 not only showed improved cellular activity, but also showed improved selectivity for other JAKs in a cellular environment with high concentration of ATP. In contrast, nonselective tofacitinib did not exhibit apparent selectivity to inhibit phosphorylation of the downstream substrate stimulated by the three cytokines.

(5) Cell washout experiment

To further demonstrate that compound 32 is covalently bound to JAK3 in cells, a cell washout experiment was performed.

The washout procedure is as follows: CTLL-2 cells were treated with the compound for 2 hours. Then, in the case of the washout group, the cells were washed 3 times with PBS. The non-washout group remained constant. Cells were then stimulated with IL-15 for 30 minutes, lysed, and standard Western blots were performed. The results are shown in FIG. 9. Cells treated with the compound were extensively washed with PBS. Cells treated with compound 32 (which is covalently bound to JAK3 in cells) sustained inhibition of STAT5 phosphorylation. In comparison, the inhibitory activity of the reversible inhibitor tofacitinib and the compound NIBR3049 was almost lost after washing out. Since JAK1 does not have a cysteine in the same position as Cys909 in JAK3, compound 32 will have little potential to interfere with JAK1 activity after washing out with PBS. Thus, this washout experiment alleviated the effects of JAK1 and demonstrated that specific inhibition of JAK3 alone is sufficient to effectively inhibit the _{IL-15-mediated γ c cytokine receptor signaling pathway.}

(6) Inhibition of LPS-stimulated inflammatory cytokine release

In rheumatoid arthritis (RA) patients, joint erosion is consistent with an increase in inflammatory cytokines, including IL-6, IL-1β, TNF-α, and MCP-1. The release of IL-6, IL-1β, TNF-α and MCP-1 was regulated by negative feedback of the IL-10-JAKs-STAT3 signaling pathway.

LPS-induced IL-6 and TNF-α release assays were performed as follows: Frozen PBMCs (from Allsells) were thawed in RPMI1640 (Thermo Fisher) containing 10% FBS and overnight at 37°C. Recovered. The next day, the cells ^{were diluted to 1 × 10 6} cells/mL and seeded (500 μL) in a 6-well plate. Compound or DMSO (5 μL, serial dilution in DMSO) was added to the plate and incubated with cells at 37° C. for 2 h, then stimulated with LPS (5 μL, 1 μg/mL) and 5% CO _{2 at 37° C.} Incubated for 24 h. Supernatants were collected to determine IL-6 and TNF-α levels using human IL-6 or human THF-α DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions.

The IL-6 stimulated inflammatory cytokine MCP-1 release assay was performed in a similar manner. Experimental results are as shown in FIGS. 10 to 12.

In LPS-challenged PBMCs, the release of IL-6 and TNF-α was significantly inhibited by compound 32. In contrast, the nonselective inhibitor tofacitinib increased cytokine production to different degrees due to inhibition of IL-10-JAKs-STAT3 negative feedback signaling due to inhibition of tofacitinib on JAK1. Compound 32 sustained IL-10 signaling pathway function due to its selective inhibition on JAK3. Neither compound 32 nor tofacitinib inhibited IL-6 induced MCP-1 release (which is not mediated by JAK), which means that these two compounds modulate the release of inflammatory cytokines through the JAK-STAT signaling pathway. Is to represent. These results demonstrated that compound 32 plays an important role in modulating inflammatory cytokine release by selectively inhibiting JAK3 and inhibiting the JAK3-STAT signaling pathway.

(7) Pharmacokinetic evaluation

The pharmacokinetic (PK) properties of Compound 32 in mice were evaluated after intravenous and oral administration.

For the in vivo PK study, male ICR mice (n = 3) were fasted overnight and compound 32 received either as an intravenous dose (2 mg/kg) or via oral gavage (5 mg/kg). Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, and 24 h (iv) and 0.25, 0.5, 1, 2, 4, 8, and 24 h (po). Plasma samples were deproteinized with acetonitrile containing an internal standard. After centrifugation at 4° C., the supernatant was collected for LC/MS/MS analysis.

PK was determined by analysis of plasma concentration at the indicated time points. The results are shown in the table below. Data represent the mean concentration in plasma (n=3) after a single 2.0 mg/kg intravenous administration and 5 mg/kg oral administration.

Table: PK study of compound 32 in ICR mice

Immediately after 5 mg/kg oral delivery, Compound 32 _{exhibited a PK profile with a half-life of 1.66 h (t 1/2} ), an area under the curve (AUC) of 608 ng·hr/mL, and a moderate oral bioavailability of 24.4%. Showed. These potent PK properties suggested that Compound 32 may be an oral inhibitor or probe for further pharmacokinetic studies and biological function exploration in animals.

It should be understood that the above embodiments are for illustrative purposes only and do not constitute any limitation to the scope of protection of the present invention. The scope of protection of the present invention is determined by the appended claims, including not only the literal interpretation of the technical solutions in the claims, but also equivalents of the technical solutions in the claims. For example, stable isotope substitutes for compounds are also included within the scope of protection of the present invention.

Claims (8)

A compound of formula I, or a pharmaceutically acceptable salt thereof.

here,
Rh is H or methyl, preferably H;
Rg is CH, -C-Rf or N, preferably CH;
Rf is preferably a substituent selected from methyl or halogen (eg F, Cl, Br or I);
m is 0, 1, 2 or 3, preferably 0 or 1, more preferably 0;
Re is a tertiary amine cation (-N+R' ₃ , wherein R'is _{independently selected from H and C 1} -C ₆ alkyl), nitro (-NO ₂ ), trihalomethyl (-CX ₃ , X= F, Cl, Br or I), halogen (such as F, Cl, Br and I), formyl (-CHO), acyl (-CO-C _1-4 alkyl), carboxyl (-COOH), cyano ( -CN), and an electron-withdrawing group selected from the group consisting of a _{sulfonic acid group (-SO 3 H);}
Rd is, for example, alkenyl or alkynyl having 2, 3, 4, 5 or 6 carbon atoms;
Ra, Rb and Rc are selected from the following combinations:
(1) Rb is C ₁ -C ₄ alkylene (e.g. C ₁ -C ₃ alkylene, such as methylene, ethylidene, 1,3-propylidene),
Ra and Rc are hydrogen or C ₁ -C ₆ alkyl (eg C ₁ -C ₄ alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl);
(2) Rb is C ₁ -C ₄ alkylene (e.g. C ₁ -C ₃ alkylene, such as methylene, ethylidene, 1,3-propylidene),
Ra and Rc are attached together to form C ₂ -C ₄ alkylene (eg C ₂ -C ₃ alkylene such as ethylidene, 1,3-propylidene);
(3) Ra is hydrogen or C ₁ -C ₆ alkyl (e.g. C ₁ -C ₄ alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl),
Rb and Rc together with the N atom to which they are attached form an N atom containing 5- or 6-membered saturated heterocyclic ring;
(4) Rc is hydrogen or C ₁ -C ₆ alkyl (e.g. C ₁ -C ₄ alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl),
Ra and Rb together with the N atom to which they are attached form an N atom containing 5- or 6-membered saturated heterocyclic ring. The method of claim 1, wherein -N(Ra)-Rb-N(Rc)- is

Or a pharmaceutically acceptable salt thereof.

The compound according to claim 1 or 2, wherein Rh is H, Rg is CH, and m is 0, or a pharmaceutically acceptable salt thereof. The compound according to any one of claims 1 to 3, wherein Re is -NO ₂ or a pharmaceutically acceptable salt thereof. The compound according to any one of claims 1 to 4, wherein Rd is vinyl, or a pharmaceutically acceptable salt thereof. A compound of formula II or a pharmaceutically acceptable salt thereof:

. A compound of formula I according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a compound of formula II according to claim 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Pharmaceutical composition comprising. In the manufacture of a medicament for the treatment of inflammation, such as rheumatoid arthritis, a compound of formula I according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, a compound of formula II according to claim 6, or a Use of a pharmaceutically acceptable salt, or a pharmaceutical composition according to claim 3.

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